A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples
- PMID: 17097714
- DOI: 10.1016/j.watres.2006.10.003
A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples
Abstract
A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (<200 N. fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.
Similar articles
-
Detection of Naegleria spp. and Naegleria fowleri: a comparison of flagellation tests, ELISA and PCR.Water Sci Technol. 2003;47(3):117-22. Water Sci Technol. 2003. PMID: 12639015
-
A real-time PCR diagnostic method for detection of Naegleria fowleri.Exp Parasitol. 2010 Sep;126(1):37-41. doi: 10.1016/j.exppara.2009.11.001. Epub 2009 Nov 15. Exp Parasitol. 2010. PMID: 19919836
-
Comparison of next-generation droplet digital PCR with quantitative PCR for enumeration of Naegleria fowleri in environmental water and clinical samples.Lett Appl Microbiol. 2018 Oct;67(4):322-328. doi: 10.1111/lam.13051. Epub 2018 Aug 22. Lett Appl Microbiol. 2018. PMID: 30007064
-
Recent advances in quantitative PCR (qPCR) applications in food microbiology.Food Microbiol. 2011 Aug;28(5):848-61. doi: 10.1016/j.fm.2011.02.008. Epub 2011 Apr 1. Food Microbiol. 2011. PMID: 21569926 Review.
-
Distribution of pathogenic Naegleria spp in Thailand.Southeast Asian J Trop Med Public Health. 2001;32 Suppl 2:172-8. Southeast Asian J Trop Med Public Health. 2001. PMID: 12041585 Review.
Cited by
-
Occurrence and Control of Legionella in Recycled Water Systems.Pathogens. 2015 Jul 1;4(3):470-502. doi: 10.3390/pathogens4030470. Pathogens. 2015. PMID: 26140674 Free PMC article. Review.
-
Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform.Appl Environ Microbiol. 2008 Jun;74(12):3831-8. doi: 10.1128/AEM.02743-07. Epub 2008 Apr 18. Appl Environ Microbiol. 2008. PMID: 18424532 Free PMC article.
-
Survey for the presence of specific free-living amoebae in cooling waters from Belgian power plants.Parasitol Res. 2007 May;100(6):1249-56. doi: 10.1007/s00436-006-0399-1. Epub 2006 Dec 21. Parasitol Res. 2007. PMID: 17186275
-
Noninvasive diagnostic biomarkers, genomic profiling, and advanced microscopic imaging in the early detection and characterization of Naegleria fowleri infections leading to primary amebic meningoencephalitis (PAM).Ann Med Surg (Lond). 2024 Feb 28;86(4):2032-2048. doi: 10.1097/MS9.0000000000001843. eCollection 2024 Apr. Ann Med Surg (Lond). 2024. PMID: 38576920 Free PMC article. Review.
-
Development of a rapid, simple method for detecting Naegleria fowleri visually in water samples by loop-mediated isothermal amplification (LAMP).PLoS One. 2015 Mar 30;10(3):e0120997. doi: 10.1371/journal.pone.0120997. eCollection 2015. PLoS One. 2015. PMID: 25822175 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources